多環芳香烴受體對組蛋白乙醯化修飾之探討

The Role of Aryl Hydrocarbon Receptor in Acetylation Modification of Histone

梁哲睿(Che-Jui Liang)

指導教授 ： 康照洲

國立臺灣大學/醫學院/毒理學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201700155

摘要

多環芳香烴受體 (aryl hydrocarbon receptor, AhR) 屬 basic-helix-loop-helix (bHLH)/PAS 家族，此家族參與許多生理反應，像是生理週期、代謝及缺氧狀態下的壓力反應。AhR 可以調解許多藥理及毒理效應，包含誘導藥物代謝酵素 CYP1A1、免疫抑制、腫瘤促進。AhR 主要分布於細胞質並與 molecular chaperone complex (Hsp90/XAP2/p23) 結合，當受到外來物質，如：PAHs 的刺激，AhR 活化後會進入細胞核內與芳香烴核轉位蛋白 (aryl hydrocarbon nuclear translocator, ARNT) 形成複合體，此複合體會結合在 XRE/DRE 片段上，進行下游基因的轉錄。附基因體機制包括 DNA 甲基化 (DNA Methylation)、組蛋白之間轉譯後修飾的改變 (post-translational modification of histone proteins) 以及微小 RNA 的表現 (microRNA expression)。組蛋白的轉譯後修飾是附基因體調控中很重要的調控機制之一，常見的轉譯後修飾有乙醯化 (acetlaytion)、甲基化 (methylation)。這些修飾會影響組蛋白間緊密程度，進而影響基因表現。組蛋白間的乙醯化會受到兩種互相結抗的酵素所調控，分別為組蛋白乙醯基轉移酶 (histone acetyltransferase, HATs) 及組蛋白去乙醯基酶 (histone deacetylases, HDACs)。近年來研究發現癌症的發生與進展也和附基因體的失常有關，已有文獻報導 HDAC 過度表現會抑制腫瘤抑制基因 p53 的表現，亦會引起缺氧誘導因子-1 的活化進而誘發血管新生。另一方面，文獻也指出 HDAC 抑制劑，LBH589 可以抑制癌細胞的血管新生作用，過去我們也發現 AhR 與血管新生有關。所以本研究的目的為探討附基因體藥物如何影響 HDAC 並探討 AhR 蛋白與 HDAC 之間如何互相調控。本研究利用胃癌細胞株 AGS 處理 HDAC 抑制劑 (LBH589)，再利用西方點墨法分析組蛋白上的乙醯化程度，結果顯示抑制 HDAC 的活性會增加乙醯化 H4 與 H2BK5 蛋白表現，然後也看到 AhR 靜默的細胞株，乙醯化 H4 與 H2BK5 蛋白表現也明顯降低。細胞氧化壓力實驗發現，處理 LBH589 會造成細胞內氧化壓力產生。此外我們也發現 LBH589 處理不同時間及不同劑量後，AhR 及 Nrf2蛋白表現也隨之增加，具有劑量效應；且也發現隨著處理時間增加，AhR 蛋白具有時間效應，而 Nrf2 蛋白則是 3小時達到最多之後開始減少。參考先前文獻研究結果得知 HDACi 會產生氧化壓力殺死癌細胞，本實驗利用 ROS 抑制劑 NAC 發現 LBH589 所生成的 ROS 跟 AhR 及 Nrf2 蛋白的產生有關，加上使用 Nrf2 抑制劑 brusatol，證明 Nrf2 在 AhR 蛋白生成扮演重要角色。ChIP assay實驗結果發現，LBH589 會使 Nrf2 蛋白與 AhR promoter 結合。進一步利用核質分離實驗發現，LBH589 處理 24小時後會使 AhR 進核，且以 AhR 靜默組也具相同結果。HAT Activity Colorimetric Assay 及 HDAC-Glo I/II Assay 兩個活性實驗發現，A549 細胞之 AhR 靜默與控制組相比，HAT 活性沒有顯著差異；而 HDAC 活性則有明顯增加的趨勢。利用免疫沉澱法分析 AhR 與這些蛋白之間的交互作用，結果得知處理 LBH589 後，AhR 與 HDAC1、HDAC2 及 HDAC3 之間結合增加。免疫螢光染色法進一步證明 AhR 與 HDAC1、HDAC2 及 HDAC3 蛋白之間有進行交互作用。綜合以上結果，本研究發現 LBH589 會使細胞內產生 ROS 並造成 Nrf2 蛋白增加及進入細胞核與 AhR 的啟動子結合，造成 AhR 蛋白表現增加，此內生性機制之 AhR 表現會影響附基因體的調控並導致 HDAC 之負回饋抑制。

關鍵字

多環芳香烴受體 ； 組蛋白去乙醯酶抑制劑 ； NF-E2 相關因子 2 ； 組蛋白乙醯化 ； 氧化壓力 ； 組蛋白去乙醯酶

並列摘要

Aryl hydrocarbon receptor (AhR) is a ligand-activated protein, it can regulate many effects of pharmacology and toxicology. Recently, the novel function of AhR have been mentioned briefly. Current studies have revealed that changes in epigenetic regulation can alter AhR protein expression and contribute to healthy problem. Histone acetylation is one of the epigenetic modifications. This modification influence chromatin structure involved gene transcription. There are two enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), can regulate histone acetylation in this modification. However, the regulation mechanism between AhR and HDAC are not clear. In this study, we investigate the regulation between AhR and HDAC. We used gastric cancer cell lines (AGS) that treated HDACi (LBH589), assayed by Western blot to analyze acetylation of histone. Inhibition of HDACs activity can increase acetylation of histone H4 and H2BK5. Moreover, comparing between the normal AhR cells and the AhR gene knock down cells, we found that the higher acetylation of histone with higher AhR expression level. Cellular reactive oxygen species detection assay showed that LBH589 might produce ROS. Additionally, the result demonstrated that AhR and Nrf2 protein expression was up-regulation with LBH589 treatment in dose-dependent manner. Another result showed that AhR protein expression was up-regulation with LBH589 treatment in time course manner, but Nrf2 protein expression is largest in 3hours. Previous study revealed that HDACi may induce ROS to kill the cancer cell, therefore, we used ROS inhibitor, NAC, and found that increased AhR and Nrf2 protein was related to ROS with LBH589 treatment. Moreover, we utilized Nrf2 inhibitor brusatol to prove that Nrf2 is an important role in AhR production. ChIP assay proved that Nrf2 may bind with AhR promoter sequence with LBH589 treatment. Both of the HDAC-Glo I/II Assay and HAT Activity Colorimetric Assay discovered that activity of HDACs were up-regulation in shAhR A549 cells, but activity of HATs was not. Immunoprecipitation (IP) confirmed that LBH589 treatment increased AhR/HDAC1/HDAC2/HDAC3 complex. Finally, we applied the immunofluorescence staining (IF) to prove AhR can interact with HDAC1, HDAC2, and HDAC3. According to above results, we have acquired conclusion that LBH589 may induce ROS and cause Nrf2 protein bind with AhR promoter. Up-regulation of AhR may regulate epigenetic mechanism and inhibit HDACs activity.

並列關鍵字

AhR ； HDACi ； Nrf2 ； Histone acetylation ； ROS ； HDAC

參考文獻

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